Mammalian DNA ligases. Catalytic domain and size of DNA ligase I.

DNA ligase I is the major DNA ligase activity in proliferating mammalian cells. The protein has been purified to apparent homogeneity from calf thymus. It has a monomeric structure and a blocked N-terminal residue. DNA ligase I is a 125-kDa polypeptide as estimated by sodium dodecyl sulfate-gel electrophoresis and by gel chromatography under denaturing conditions, whereas hydrodynamic measurements indicate that the enzyme is an asymmetric 98-kDa protein. Immunoblotting with rabbit polyclonal antibodies to the enzyme revealed a single polypeptide of 125 kDa in freshly prepared crude cell extracts of calf thymus. Limited digestion of the purified DNA ligase I with several reagent proteolytic enzymes generated a relatively protease-resistant 85-kDa fragment. This domain retained full catalytic activity. Similar results were obtained with partially purified human DNA ligase I. The active large fragment represents the C-terminal part of the intact protein, and contains an epitope conserved between mammalian DNA ligase I and yeast and vaccinia virus DNA ligases. The function of the N-terminal region of DNA ligase I is unknown.     

Different conformational states of the purified Ca2+-ATPase of the erythrocyte plasma membrane revealed by controlled trypsin proteolysis

The purified Ca2+-pumping ATPase of the erythrocyte membrane has been exposed to trypsin at 37 degrees C, in the presence of different effectors of its activity. The control proteolytic pattern is characterized by a number of transient and of limit polypeptides (Zurini, M., Krebs, J., Penniston, J. T., and Carafoli, E. (1984) J. Biol. Chem. 259, 618-627). The effectors influence the pattern in the Mr region 90,000-76,000, which contains the calmodulin binding domain and the active site of the enzyme. In this region, polypeptides of 90, 85, 81, and 76 kDa are clearly visible in the controls. 1) Calmodulin plus Ca2+ induces the faster disappearance of the 90-kDa product and the relative accumulation of the 85-kDa with respect to the 81-kDa polypeptide. 2) Vanadate plus Mg2+ also accelerates the disappearance of the 90-kDa product. However, they induce the relative accumulation of the 81-kDa polypeptide. 3) Linoleic acid, which stimulates the activity of the enzyme to the same levels obtained with calmodulin, greatly accelerates the rate of trypsin proteolysis, causing the virtual disappearance of all polypeptides in the 90-76-kDa region. 4) The 81-kDa polypeptide has maximal ATPase activity and is insensitive to calmodulin; the 85-kDa polypeptide has lower ATPase activity and binds calmodulin, but is not stimulated (or is stimulated only negligibly) by the activator.     

The purification of a human mismatch-binding protein and identification of its associated ATPase and helicase activities.

A mismatch-binding protein has been purified an estimated 4500-fold from HeLa nuclear extracts using four different chromatographic steps. Two polypeptides of apparent molecular weight of 160,000 and 100,000 were present in the final affinity-purified fraction as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Partial proteolytic clipping of the protein-DNA complexes visualized after UV treatment indicated that the 100-kDa polypeptide is most likely a degradation product of the 160-kDa polypeptide. UV cross-linking experiments have shown that both these polypeptides bind specifically to oligonucleotide duplexes containing G/T mismatches. Direct DNA binding studies and band-shift competition assays showed that although the mismatch-binding protein binds with highest affinity to oligonucleotides containing G/T mismatches, it is also capable of binding to oligonucleotides containing other mispairs. The purified protein has an associated Mg(2+)-dependent ATPase activity, which is markedly enhanced in the presence of single-stranded DNA. A helicase capable of unwinding a 34-mer oligonucleotide, annealed to a complementary sequence in single-stranded M13, also copurified with the mismatch-binding protein. This reaction occurs in an ATP- and magnesium-dependent manner.     

Thermostable DNA polymerase from the archaebacterium Sulfolobus acidocaldarius. Purification, characterization and immunological properties

We have purified to near homogeneity a DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed a polypeptide of 100 kDa. On the basis of a Stokes radius of 4.2 nm and a sedimentation coefficient of 6 S, the purified enzyme has an estimated molecular mass of 109 kDa. These results are consistent with the enzyme being a monomer of 100 kDa. In addition a polyclonal antiserum, obtained by injection of the electroeluted 100-kDa polypeptide into a rabbit, specifically neutralized the DNA-polymerase activity. The enzyme is sensitive to both N-ethylmaleimide and 2',3'-dideoxyribosylthymine triphosphate and resistant to aphidicolin. The purified DNA polymerase has neither exonuclease nor primase activities. In our in vitro conditions, the enzyme is thermostable up to 80 degrees C and is active between 55 degrees C and 85 degrees C in the presence of activated calf-thymus DNA.     

Identification and tryptic cleavage of the catalytic core of HeLa and calf thymus DNA polymerase epsilon

DNA polymerase epsilon, formerly known as a proliferating cell nuclear antigen-independent form of DNA polymerase delta, has been shown elsewhere to be catalytically and structurally distinct from DNA polymerase delta. The catalytic activity of HeLa DNA polymerase epsilon, an enzyme consisting of greater than 200- and 55-kDa polypeptides, was assigned to the larger polypeptide by polymerase trap reaction. This catalytic polypeptide was cleaved by incubation with trypsin into two polypeptide fragments with molecular masses of 122 and 136 kDa, the former of which was relatively resistant to further proteolysis and possessed the polymerase activity. The cleavage increased the polymerase and exonuclease activities of the enzyme some 2-3-fold. DNA polymerase epsilon was also purified in a smaller 140-kDa form from calf thymus. The digestion of this form of the enzyme by trypsin also generated a 122-kDa polypeptide. These results suggest that the catalytic core of DNA polymerase epsilon is a 258-kDa polypeptide that is composed of two segments linked with a protease-sensitive area. One of the segments harbors both DNA polymerase and 3'----5' exonuclease activities. In spite of the different polypeptide structures, the catalytic properties of the HeLa enzyme, its trypsin-digested form, and the calf thymus enzyme remained essentially the same.     

Purification and characterization of a gamma-like DNA polymerase from Chlamydomonas reinhardtii

A crude in vitro system which initiates chloroplast DNA synthesis near the D-loop site mapped by electron microscopy [Wu, M., Lou, J. K., Chang, D. Y., Chang, C. H., & Nie, Z. Q. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6761-6765] consists of soluble proteins and proteins extracted from purified thylakoid membrane. In this paper, a DNA polymerase activity was purified to near homogeneity from the soluble protein fraction of this in vitro system by sequential chromatographic separations on heparin-agarose, DEAE-cellulose, and single-stranded DNA-agarose columns and sedimentation in a glycerol gradient. In the glycerol gradient, the enzyme activity sedimented at a position corresponding to a 110-kDa protein. Electrophoretic analysis of the highly purified fraction on SDS-polyacrylamide gel revealed a major polypeptide band with an apparent molecular mass of approximately 116 kDa. In situ DNA polymerase activity assay shows that the DNA polymerization function is associated with the 116-kDa band and an 80-kDa band which could be a subunit of the enzyme. Polymerization activity is inhibited by N-ethylmaleimide, ethidium bromide, and dideoxycytosine triphosphate and is relatively resistant to aphidicolin. Poly(dA).(dT)10 and gapped double-stranded DNA are preferred templates. The purified enzyme contains no exonuclease activity and can initiate DNA replication in a supercoiled plasmid DNA template containing the chloroplast DNA replication origin.     

Size heterogeneity of the largest subunit of nuclear RNA polymerase II. An immunological analysis

Antibodies raised against the 180-kDa subunit of cauliflower RNA polymerase II bind selectively to the largest subunit of RNA polymerase II purified from a variety of plant species. The selective binding of this antibody to the largest RNA polymerase II subunit has allowed us to probe for the size of this subunit in crude cell extracts, in fractions containing partially purified RNA polymerase II, and in isolated nuclei. Fractions containing RNA polymerase II were subjected to electrophoresis in the presence of sodium dodecyl sulfate, blotted onto nitrocellulose, and blots were probed with antibody. Immunoglobulin complexes were revealed with 125I-Protein A. Published purification procedures result in rapid conversion of a 220-kDa subunit to a 180-kDa polypeptide, but purification at high pH (pH 9.0) retards this proteolysis. RNA polymerase II associated with isolated nuclei is largely protected from proteolytic degradation, and a 240-kDa polypeptide as well as a 220-kDa polypeptide can be detected. These results suggest that the 180-kDa subunit of RNA polymerase II arises artificially during cell lysis and enzyme purification, and that even the 220-kDa polypeptide may be a degradation product of a 240-kDa polypeptide in plants.     

Purification and characterization of a new DNA-dependent ATPase with helicase activity from Escherichia coli

A previously unreported single-stranded DNA-dependent nucleoside 5'-triphosphatase with DNA unwinding activity has been purified from extracts of Escherichia coli lacking the F factor. Fractions of the purified enzyme contain a major polypeptide of Mr = 75,000 which contains the active site(s) for both ATP hydrolysis and helicase activity. This is consistent with the results of gel filtration chromatography which indicate a native molecular mass of 75 kDa. The 75-kDa helicase has a preference for ATP (dATP) as a substrate in the hydrolysis reaction and requires the presence of a single-stranded DNA cofactor. The helicase reaction catalyzed by the enzyme has been characterized using an in vitro strand displacement assay. The 75-kDa helicase displaces a 71-nucleotide DNA fragment in an enzyme concentration-dependent and time-dependent reaction. The helicase reaction depends on the presence of a hydrolyzable nucleoside 5'-triphosphate (NTP) suggesting that NTP hydrolysis is required for the unwinding activity. In addition, the enzyme can displace a 343-nucleotide DNA fragment albeit less efficiently. The direction of the unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The molecular size of this helicase and the direction of the unwinding reaction are similar to both helicase II and Rep protein. However, the 75-kDa helicase has been shown to be distinct from both helicase II and Rep protein using immunological, physical, and genetic criteria. The discovery of a new helicase brings the total number of helicases found in E. coli cell extracts (lacking F factor) to five.     

Purification of a site-specific endonuclease, I-Sce II, encoded by intron 4 alpha of the mitochondrial coxI gene of Saccharomyces cerevisiae

We have purified to near homogeneity a site-specific, double-stranded DNA endonuclease (I-Sce II) encoded by intron 4 alpha (aI4 alpha) of the yeast mitochondrial coxI gene. Our purification starts with a high salt extract of mitochondria isolated from a yeast strain that overproduces the enzyme because of a block in splicing of aI4 alpha. The final step of purification is an affinity column consisting of covalently bound double-stranded DNA multimers of a synthetic sequence, 5'-TTGGTCATCCAGAAGTAT-3', which contains the I-Sce II cleavage/recognition site. Typical yields of enzyme are 3-5% with a specific activity of approximately 500,000 units/mg, where 1 unit of activity cleaves 50 ng of DNA substrate/h at 30 degrees C. I-Sce II has a monomer molecular mass of 31 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Active enzyme purifies as a 55-kDa species, which we presume to be a homodimer. I-Sce II monomer comigrates with an in vivo synthesized mitochondrial translation product made in the strain that overproduces the enzyme. We conclude that I-Sce II is derived by proteolytic processing of a precursor polypeptide, p62, encoded by an in-frame fusion of coxI exons 1-4 with the downstream aI4 alpha reading frame. I-Sce II is most active at pH 7.5 and at 20-30 degrees C. Endonuclease activity is sensitive to salt and is dependent upon Mg2+ or Mn2+, but is unaffected by inclusion of ATP or GTP. I-Sce II is the first intron-encoded protein to be purified and characterized from yeast mitochondria.     

Mammalian DNA ligases. Biosynthesis and intracellular localization of DNA ligase I

Mammalian DNA ligase I is presumed to act in DNA replication. Rabbit antibodies against the homogeneous enzyme from calf thymus inhibited DNA ligase I activity and consistently recognized a single polypeptide of 125 kDa when cells from an established bovine kidney cell line (MDBK) were lysed rapidly by a variety of procedures and subjected to immunoblotting analysis. After biosynthetic labeling of MDBK cells with [35S]methionine, immunoprecipitation experiments revealed a polypeptide of 125 kDa that did not appear when purified calf thymus DNA ligase I was used in competition. A 125-kDa polypeptide was adenylated when immunoprecipitated protein from MDBK cells was incubated with [alpha-32P]ATP. Thus, the apparent molecular mass of the initial translation product is identical or nearly so to that of the purified enzyme. The half-life of the protein is 7 h as determined by pulse-chase experiments in asynchronous MDBK cells. Immunocytochemistry and indirect immunofluorescence experiments showed that DNA ligase I is localized to cell nuclei.     

Isolation of the DNA polymerase alpha core enzyme from mouse cells

DNA polymerase alpha has been purified from mouse hybridoma cells approximately 30,000-fold using a combination of conventional and high performance liquid chromatography. In contrast to previous characterizations of mammalian DNA polymerase alpha, this enzyme has a single high molecular mass polypeptide (185 kDa) in tight association with a 68-kDa polypeptide and this structure appears to be the core DNA polymerase of the mouse cells. The biochemically purified enzyme, with a specific activity of approximately 200,000 units/mg protein, has an estimated molecular mass by gel filtration chromatography of 240 kDa and sedimentation value of 9 S, consistent with the enzyme being a heterodimer of 185 and 68 kDa. The enzyme is sensitive to both N-ethylmaleimide and aphidicolin and insensitive to ddTTP. Using an activated DNA template, the apparent Km values for the deoxynucleotide triphosphates are approximately 0.5-1 microM. The purified DNA polymerase has neither exonuclease nor primase activities and is the predominant DNA polymerase alpha activity in the mouse cells.     

Dictyostelium myosin light chain kinase. Purification and characterization

A Dictyostelium myosin light chain kinase has been purified approximately 15,000-fold to near homogeneity. The purified kinase is a single polypeptide of approximately 34 kDa that phosphorylates only the 18-kDa Dictyostelium myosin regulatory light chain and itself among substrates tested. The enzyme was purified largely by ammonium sulfate fractionation and hydrophobic (butyl) interaction chromatography. Analysis using polyclonal antibodies raised against the purified 34-kDa protein confirms that this protein is responsible for myosin light chain kinase activity. Protein microsequence of the 34-kDa protein reveals conserved protein kinase sequences. The purified Dictyostelium myosin light chain kinase exhibits a Km for Dictyostelium myosin of 4 microM and a Vmax of 8 nmol/min/mg. Unlike other characterized myosin light chain kinases, this enzyme is not regulated by calcium/calmodulin. Western blot analysis demonstrates that the purified kinase is not a proteolytic fragment that has lost calcium/calmodulin regulation. The Dictyostelium myosin light chain kinase activity is not directly regulated by cyclic nucleotides. However, this kinase undergoes an intramolecular autophosphorylation that activates the enzyme.     

Purification and characterization of a DNA polymerase from the archaebacterium Thermoplasma acidophilum

A thermophilic DNA polymerase has been purified to near homogeneity from the archaebacterium Thermoplasma acidophilum. Analysis of the purified enzyme by sodium dodecyl sulfate gel electrophoresis revealed a single polypeptide of 88 kDa which co-sediments with the DNA polymerase activity on sucrose gradients. Combination of sedimentation and gel filtration analyses indicates that this DNA polymerase is an 88-kDa monomeric enzyme in its native form. The DNA polymerase is resistant to aphidicolin, slightly sensitive to 2',3'-dideoxyribosylthymine triphosphate and inhibited by N-ethylmaleimide when preincubation with this reagent is performed at 65 degrees C. We find that a 3'----5' exonuclease activity is associated with the purified DNA polymerase; the two activities of the enzyme are optimal at 65 degrees C but the exonuclease activity is active in a broader range of lower temperatures and is more thermostable than the DNA polymerase activity.     

Identification of a viral protein involved in post-translational maturation of the encephalomyocarditis virus capsid precursor.

Translation of encephalomyocarditis virus RNA in a cell-free system from uninfected Krebs ascites cells results in the synthesis of a major polypeptide product with a molecular weight of approximately 112,000. In contrast, when the viral RNA is translated in a cell-free system from virus-infected cells, this polypeptide is absent and the largest polypeptide produced has a molecular weight of about 100,000. This latter polypeptide comigrates on sodium dodecyl sulfate-gels with in vivo virus capsid precursor A, and the two have identical patterns of CNBr-generated peptides. A polypeptide having a molecular weight of 12,500 is also a major translation product in the system from infected cells (but not from uninfected cells). This polypeptide appears to be generated by cleavage of the NH-2-terminal portion of the viral RNA-dependent polypeptides by a proteolytic activity present in the infected cell-free system. This proteolytic activity copurifies with the 23,000-molecular weight viral capsid protein gamma, found in infected cells, through chromatography on DEAE-cellulose and cellulose phosphate. This suggests that gamma is itself a proteolytic enzyme involved in maturation of the viral capsid precursor.     

Purification and characterization of a novel ATP-independent type I DNA topoisomerase from a marine methylotroph

DNA topoisomerase is involved in DNA repair and replication. In this study, a novel ATP-independent 30-kDa type I DNA topoisomerase was purified and characterized from a marine methylotroph, Methylophaga sp. strain 3. The purified enzyme composed of a single polypeptide was active over a broad range of temperature and pH. The enzyme was able to relax only negatively supercoiled DNA. Mg(2+) was required for its relaxation activity, while ATP gave no effect. The enzyme was clearly inhibited by camptothecin, ethidium bromide, and single-stranded DNA, but not by nalidixic acid and etoposide. Interestingly, the purified enzyme showed Mn(2+)-activated endonuclease activity on supercoiled DNA. The N-terminal sequence of the purified enzyme showed no homology with those of other type I enzymes. These results suggest that the purified enzyme is an ATP-independent type I DNA topoisomerase that has, for the first time, been characterized from a marine methylotroph.     

Thymidine kinase from Tetrahymena thermophila. Purification and immunological analysis

Thymidine kinase is an enzyme involved in DNA precursor metabolism and DNA replication. The synthesis of this enzyme is highly regulated during the cell cycle and the activity of the enzyme is also regulated by feedback inhibition. Genes encoding thymidine kinase have been extremely useful as selectable markers for introducing DNA into a number of cells. In order to study cell cycle regulation of thymidine kinase, the gene which encodes this enzyme, as well as aspects of DNA replication in the ciliated protozoan Tetrahymena thermophila, we have purified thymidine kinase from Tetrahymena. Two forms of thymidine kinase with native molecular masses of 59 kDa and 80 kDa have been identified and purified 6800- and 4600-fold, respectively. The 59-kDa enzyme, a homodimer of 30-kDa subunits, has been purified to near homogeneity and polyclonal antibodies have been raised against the 30-kDa subunit. Serological studies indicate that the two enzymes are antigenically distinct. The antibody against the Tetrahymena protein cross-reacts with a polypeptide in Chinese hamster ovary (CHO) cell extracts of 26 kDa which corresponds to the reported size of Chinese hamster thymidine kinase protein.     

Characterization of a human coronavirus (strain 229E) 3C-like proteinase activity.

The RNA polymerase gene of human coronavirus (HCV) 229E encodes a large polyprotein that contains domains with motifs characteristic of both papain-like cysteine proteinases and proteinases with homology to the 3C proteinase of picornaviruses. In this study, we have, first, expressed the putative HCV 229E 3C-like proteinase domain as part of a beta-galactosidase fusion protein in Escherichia coli and have shown that the expressed protein has proteolytic activity. The substitution of one amino acid within the predicted proteinase domain (His-3006-->Asp-3006) abolishes, or at least significantly reduces, this activity. Amino-terminal sequence analysis of a purified, 34-kDa cleavage product shows that the bacterial fusion protein is cleaved at the dipeptide Gln-2965-Ala-2966, which is the predicted amino-terminal end of the putative 3C-like proteinase domain. Second, we have confirmed the proteolytic activity of a bacterially expressed polypeptide with the amino acid sequence of the predicted HCV 229E 3C-like proteinase by trans cleavage of an in vitro translated polypeptide encoded within open reading frame 1b of the RNA polymerase gene. Finally, using fusion protein-specific antiserum, we have identified a 34-kDa, 3C-like proteinase polypeptide in HCV 229E-infected MRC-5 cells. This polypeptide can be detected as early as 3 to 5 h postinfection but is present in the infected cell in very low amounts. These data contribute to the characterization of the 3C-like proteinase activity of HCV 229E.     

Photoaffinity labeling of glucosyltransferase of the dolichol cycle from rat mammary gland

UDP-Glc:dolichol phosphate glucosyltransferase from lactating rat mammary gland has been partially purified by a combination of (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography on DEAE-TSK, and affinity chromatography. The partially purified enzyme exhibited several protein bands when examined by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions; among these, a 35-kDa polypeptide was quite prominent and appeared to be enriched during purification. Photoaffinity labeling of the partially purified enzyme preparation with 5-azido-[beta-32P]UDP-Glc identified a 35-kDa polypeptide. Labeling of a solubilized enzyme preparation from crude and stripped microsomes also revealed a 35-kDa band on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Photoinsertion of the probe in this polypeptide is enhanced by the presence of dolichol phosphate and Mg2+. Competition studies with UDP-Glc, UDP-glucuronic acid, other sugar nucleotides, and Glc-1-phosphate provide evidence to validate the specificity of photoaffinity labeling. These studies indicate that this 35-kDa polypeptide is involved in the synthesis of dolichol-P-Glc in rat mammary tissue. The possibility that this polypeptide may represent glucosyltransferase has been discussed.     

Expression of active rat DNA polymerase beta in Escherichia coli

A recombinant plasmid for expression of rat DNA polymerase beta was constructed in a plasmid/phage chimeric vector, pUC118, by an oligonucleotide-directed mutagenesis technique. The insert contained a 1005 bp coding sequence for the whole rat DNA polymerase beta. The recombinant plasmid was designed to use the regulatory sequence of Escherichia coli lac operon and the initiation ATG codon for beta-galactosidase as those for DNA polymerase beta. The recombinant clone, JMp beta 5, obtained by transfection of E. coli JM109 with the plasmid, produced high levels of DNA polymerase activity and a 40-kDa polypeptide that were not detected in JM109 cell extract. Inducing this recombinant E. coli with isopropyl beta-thiogalactopyranoside (IPTG) yielded amounts of 40-kDa polypeptide as high as 19.3% of total protein. Another recombinant clone, JMp beta 2-1, which was constructed by an oligonucleotide-directed mutagenesis to use the second ATG codon for the initiation codon, thus deleting the first 17 amino acid residues from the amino terminus, produced neither high DNA polymerase activity nor the 40-kDa polypeptide. The evidence suggests that this amino-terminal structure is important for stability of this enzyme in E. coli. The DNA polymerase was purified to homogeneity from the IPTG-induced JMp beta 5 cells by fewer steps than the procedure for purification of DNA polymerase beta from animal cells. The properties of this enzyme in activity, chromatographic behavior, size, antigenicity, and also lack of associated nuclease activity were indistinguishable from those of DNA polymerase beta purified from rat cells, indicating the identity of the overproduced DNA polymerase in the JMp beta 5 and the rat DNA polymerase beta.